This research proposal is focused on elucidating the nature of the interaction of parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) with their shared human receptor using an approach which integrates chemistry, molecular biology and pharmacology. The PTH/PTHrP hormone-receptor system is rich in structural diversity and possesses features providing an unusually strong foundation for structure--activity studies; a broad array of well-characterized hormone analogs (spanning the full spectrum of pharmacologic properties from agonists of enhanced activity to partial agonists to potent pure antagonists) and the convergence of two independent endogenous hormones on the same human receptor. Thus far, PTH/PTHrP analog design has been performed without benefit of knowledge regarding receptor structure or details of the molecular interaction between hormone and receptor. In the proposed studies, differences in the interaction with receptor of hormone agonists, antagonists and partial agonists will be defined at the level of amino acid-to-amino acid contact points between ligand and cloned expressed human receptor. A novel set of radiolabeled PTH/PTHrP analogs incorporating a photoreactive group will be designed, synthesized, and evaluated in a battery of in vitro bioassays. Photochemical cross- linking studies will be performed. Radiolabeled fragments of hormone- receptor conjugates will be isolated and sequenced. By moving the arylketone cross-linking moiety to different positions along the ligand, a map of the binding surfaces of hormone and receptor will be obtained. Putative points of interaction will be confirmed by substituting specific residues in the ligand (by chemical synthesis) or in the receptor (by site-directed mutagenesis). After an initial round of cross-linking and microsequencing investigations using agonist, antagonist and partial agonist ligands, a new set of analogs based on the first set of insights will be designed. These novel ligands, and ligands based on subsequent rounds of cross-linking will be used in an iterative manner to steadily advance understanding of hormone-receptor interactions. In addition, structural requirements for signal transduction and receptor desensitization may be elucidated. All analogs will be evaluated in a battery of in vitro bioassays. The clinical importance of hyperparathyroidism and hypercalcemia of malignancy, and the growing interest in the therapeutic potential of PTH/PTHrP agonist analogs for treating osteoporosis provide impetus for these investigations. Furthermore, insights into fundamental principles regarding the general nature of peptide hormone--receptor interactions should emerge.